Project Summary Myelination of the central nervous system (CNS) by oligodendrocytes (OLs) is essential for the development and function of the CNS. Myelin develops in the CNS as OLs differentiate into the myelin-forming mature phenotype. The transcriptional regulatory network governing OL differentiation remains incompletely understood. To address this fundamental issue, an innovative CRISPRi (CRISPR interference) screen has been developed that can identify novel transcription factors that are important for OL differentiation. In this screen, the expression of a candidate transcription factor is knocked down by CRISPRi, a potent gene knockdown technique, and its impact on the differentiation of primary mouse OPCs is measured by the reporter activity of Rffl, a well-characterized Myrf luciferase reporter. Myrf is a key OL transcription factor that is indispensable for OL differentiation and CNS myelination. Myrf expression marks the onset of OL differentiation. Rffl is an OL enhancer in the Rffl locus that is bound and activated by Myrf in differentiating OLs. The activation of Rffl by Myrf is highly specific and sensitive. These features make Rffl an ideal luciferase reporter for OL differentiation, making it possible to read out OL differentiation by a simple luciferase assay. Capitalizing on CRISPRi and Rffl as well as in-house optimized protocols for primary mouse OPCs, the CRISPRi screen has so far tested 87 transcription factors that are highly expressed in OL lineage cells. Well- characterized OL transcription factors such as YY1, Sox10, and Myrf came out positive. The screen has also uncovered 8 novel transcription factors that are required for OL differentiation, including Rnf10. These results demonstrate the validity and effectiveness of the CRISPRi screen, justifying its further application (Aim I). Rnf10 is a transcriptional regulator that plays a pivotal role in neuronal differentiation and synaptonuclear communication. However, nothing is known about its role in OL lineage cells. Since the preliminary data strongly suggest a crucial role of Rnf10 in OL differentiation, its role in CNS myelination will be determined (Aim II).